Contribution of Extracellular Glutamine as An Anaplerotic Substrate to Neuronal Metabolism: A Re-Evaluation by Multinuclear NMR Spectroscopy in Primary Cultured Neurons

Multinuclear NMR spectroscopy is used to investigate the effect of glutamine on neuronal glucose metabolism. Primary neurons were incubated with [1-¹³C]glucose in the absence or presence of glutamine (2 mM) and/or NH₄Cl (5 mM). After ammonia-treatment, the concentrations of high-energy phosphates decreased up to 84% of control, which was aggravated in glutamine-containing medium (up to 42% of control). These effects could not be attributed to changes in mitochondrial glucose oxidation. Withdrawal of glutamine decreased amino acid concentrations, e.g. of glutamate to 53%, but also considerably lessened the ¹³C enrichment in [4-¹³C]glutamate to 8.3% of control, and decreased the ¹³C-enrichment in acetyl-CoA entering the Krebs cycle (P<0.001). Thus, although glutamine is potent in replenishing neuronal glutamate stores, glutamate formation is mainly attributed to its de novo synthesis from glucose. Furthermore, mitochondrial glucose metabolism strongly depends on the supply of carbons from glutamine, indicating that exogenous glutamine is a well-suited substrate to replenish neuronal Krebs cycle intermediates.     

Evidence for the role of intracellular glutamine level in the regulation of glutamine uptake in the cyanobacteriumAnabaena 7120

The role of intracellular glutamine concentration in the regulation of¹⁴C-glutamine uptake was studied in a diazotrophic cyanobacteriumAnabaena 7120. The uptake pattern was found to be biphasic, consisting of a rapid first phase lasting up to 60 s followed by a slower second phase. Azaserine, which could not inhibit in vitro and in vivo glutamine synthetase (GS) activity effectively, inhibited the¹⁴C-glutamine uptake. Glutamine uptake was also not significantly affected when glutamate, methylglutamate, aspartate, arginine, lysine, hydroxylysine, ornithine, and GS inhibitor,L-methionine-DL-sulfoximine (MSX) were simultaneously available during uptake assay, suggesting that glutamine uptake takes place via a general amino acid permease which does not, however, transport basic and acidic amino acids. The azaserine-treated cells had increased and decreased levels of glutamine and glutamate, respectively, suggesting that the increased intracellular glutamine level is responsible for the inhibition of¹⁴C-glutamine uptake and provides evidence here for the role of an intracellular glutamine pool in the regulation of¹⁴C-glutamine uptake inAnabaena 7120.     

Intraventricular cholecystokinin-octapeptide produces hyperglycemia in rats

The effect of intraventricular cholecystokinin-octapeptide (CCK-8) on blood glucose was evaluated. Intraventricular CCK-8 in rats produces hyperglycemia. The highest dose of CCK-8 (250 ng) increased plasma glucagon levels but at lower doses (2.5 and 25 ng) increases in glucose occurred without alteration in the glucagon levels. None of the doses of CCK-8 altered insulin levels. Using ¹⁴C-glucose tracer we showed that the hyperglycemia produced by CCK-8 was not due to alterations in glucose clearance.     

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

Summary High yields of human hepatocytes (up to 23×10⁶ viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10⁻⁸ M insulin, and 10⁻⁸ M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg⁻¹·min⁻¹) similar to that reported for human liver. Insulin at 10⁻⁸ M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10⁻⁹ M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α₁-antitrypsin, α₁-antichymotrypsin, α₁-acid glycoprotein, haptoglobin, α₂-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10⁻⁹ M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg⁻¹ cell protein·min⁻¹, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg⁻¹). The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.     

The time course of glucagon action on the utilization of [U-14C]palmitate by isolated hepatocytes

The time course of glucagon action on the utilization of [U-¹⁴C]palmitate by isolated hepatocytes was studied. Ten minutes incubation of the cells after hormone addition was required in order to observe increased oxidation and decreased esterification of the labeled palmitate. The acid-soluble, labeled oxidation products could be separated into two main fractions, glucose and ketone bodies. Initially, glucagon directed the flux of radioactivity toward glucose and CO₂. After prolonged incubation in the presence of glucagon, labeled ketone bodies, as well as labeled glucose and ¹⁴CO₂, were increased. This effect was most marked as regards glucose. The results indicate that glucagon induces a rapidly onset stimulation of the rates of Krebs cycle and gluconeogenesis, while increased oxidation and decreased esterification of palmitate are time-delayed corresponding to the establishment of a lower level of glycerophosphate. About 10% of the glucose carbon formed by gluconeogenesis originated from the fatty acid when cells from fasted rats were incubated in the presence of alanine and [U-¹⁴C]palmitate.     

Regulation of hormone biosynthesis in cultured islet cells from anglerfish

Summary The effects of glucose and arginine on islet hormone biosynthesis were investigated using primary cell cultures prepared from islets of the anglerfish (Lophius americanus). After dispersion under sterile conditions, islet cells were maintained at 23° C in medium containing RPMI 1640 with Hanks' buffer, pH 7.5, modified by the adjustment of glucose (to 0.56 or 5.6 mM) and arginine (to 0.1, 1.15, or 10 mM) with the addition of 10% fetal bovine serum (dialyzed, heat inactivated) and penicillin/streptomycin. After 48 h, media were replaced by incorporation media containing [¹⁴C]isoleucine and [³H]tryptophan and incubated for an additional 8 h under otherwise identical conditions. Culture samples (cells plus media) were extracted, desalted, and gel filtered to identify and quantitate [¹⁴C]insulin, [³H]glucagon(s) plus [³H]somatostatin-28, and [³H]somatostatin-14 were In some experiments, [¹⁴C]insulin, [³H]glucagon(s), [³H]somatostatin-28, and [³H]somatostatin-14 were separated by high performance liquid chromatography. Raising the medium glucose from 0.56 (control) to 5.6 mM resulted in an augmentation in incorporation of [¹⁴C]isoleucine into insulin and an augmentation of [³H]tryptophan into glucagon(s) and somatostatin-14, but no change in incorporation of [³H]tryptophan into somatostatin-28. Raising the concentration of arginine from 0.1 to 1.15 or 10 mM resulted in a dose-dependent inhibition of labeled amino acid incorporation into all hormones except somatostatin-28. The results demonstrate the usefulness of the culture system for studying the modulation of hormone biosynthesis in anglerfish islet cells. This work was supported by Grants AM 16921 and AM 26378 from the National Institutes of Health, Bethesda, MD.     

Role of glucose and insulin in regulating glycogen synthase and phosphorylase activities in rainbow trout hepatocytes

This study, using ¹³C nuclear magnetic resonance spectroscopy showed enrichment of glycogen carbon (C1) from ¹³C-labelled (C1) glucose indicating a direct pathway for glycogen synthesis from glucose in rainbow trout (Oncorhynchus mykiss) hepatocytes. There was a direct relationship between hepatocyte glycogen content and total glycogen synthase, total glycogen phosphorylase and glycogen phosphorylase a activities, whereas the relationship was inverse between glycogen content and % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio. Incubation of hepatocytes with glucose (3 or 10 mmol·1^-1) did not modify either glycogen synthase or glycogen phosphorylase activities. Insulin (porcine, 10^-8 mol·1^-1) in the medium significantly decreased total glycogen phosphorylase and glycogen phosphorylase a activities, but had no significant effect on glycogen synthase activities when compared to the controls (absence of insulin). In the presence of 10 mmol·1^-1 glucose, insulin increased % glycogen synthase a and decreased % glycogen phosphorylase a activities in trout hepatocytes. Also, the effect of insulin on the activities of % glycogen synthase a and glycogen synthase a/glycogen phosphorylase a ratio were more pronounced at low than at high hepatocyte glycogen content. The results indicate that in trout hepatocytes both the glycogen synthetic and breakdown pathways are active concurrently in vitro and any subtle alterations in the phosphorylase to synthase ratio may determine the hepatic glycogen content. Insulin plays an important role in the regulation of glycogen metabolism in rainbow trout hepatocytes. The effect of insulin on hepatocyte glycogen content may be under the control of several factors, including plasma glucose concentration and hepatocyte glycogen content.     

Enhanced production and isotope enrichment of recombinant glycoproteins produced in cultured mammalian cells

NMR studies of post-translationally modified proteins are complicated by the lack of an efficient method to produce isotope enriched recombinant proteins in cultured mammalian cells. We show that reducing the glucose concentration and substituting glutamate for glutamine in serum-free medium increased cell viability while simultaneously increasing recombinant protein yield and the enrichment of non-essential amino acids compared to culture in unmodified, serum-free medium. Adding dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, further improves cell viability, recombinant protein yield, and isotope enrichment. We demonstrate the method by producing partially enriched recombinant Thy1 glycoprotein from Lec1 Chinese hamster ovary (CHO) cells using U-¹³C-glucose and ¹⁵N-glutamate as labeled precursors. This study suggests that uniformly ¹⁵N,¹³C-labeled recombinant proteins may be produced in cultured mammalian cells starting from a mixture of labeled essential amino acids, glucose, and glutamate.     

Cyclic AMP and cyclic GMP as the respective mediators of the intracycle stimulation of DNA synthesis and mitosis induced by glucagon and insulin in primary neonatal rat hepatocytes

Within a wide range of concentrations ($\text{i.e.}$, from 10^?15 to 10^?8 mole/1), equimolar mixtures of dibutyryl-cyclic AMP and dibutyryl-cyclic GMP or of glucagon and dibutyryl-cyclic GMP or of insulin and dibutyryl-cyclic AMP faithfully mimicked the stimulation of DNA-synthetic and mitotic activities elicited by equimolar associations of glucagon and insulin in 4-to-5-day-old neonatal rat hepatocytes in primary tissue culture. These observations strongly suggest that the intracycle, growth-promoting effects of the two pancreatic hormones are mediated via both purine cyclic nucleotides in the neonatal rat hepatocytes.     

N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway

Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5^−/− mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5^−/− cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5^−/− hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor.     

Glucose Utilization by Euglena gracilis var. bacillaris at Higher pH*

SYNOPSIS. Euglena gracilis var. bacillaris is able to grow luxuriantly on glucose in a mineral salts medium at pH 6.8–7.1 following an adaptation period of about 200 hr. If adapted cells are used as an inoculum or if 0.1% glycine is included in the medium, the lag is shortened to 70–100 hr. Inclusion of 0.1% acetate in the medium produces a diphasic growth pattern, with acetate being metabolized first, followed by the later (about 400 hr) utilization of the glucose. Glucose utilization was found to be sensitive to pH as compared to growth on ethyl alcohol. However, glycine partially overcame this sensitivity. Glycine is maximally stimulatory with regard to growth on glucose at pH 7.0 at a concentration of 0.03%, thus suggesting that it functions as a sparking substance. Glycine markedly stimulates the assimilation of ¹⁴C-glucose. A number of Krebs cycle acids and amino acids were also found to stimulate ¹⁴C-glucose assimilation at neutral pH. Adaptation to glucose utilization at neutral pH was due to the appearance of mutants able to grow more rapidly under these conditions. The nature of this mutation was not determined.     

The effects of carbon-13 incorporation into preimplantation mouse embryos on development before and after implantation

Preimplantation mouse embryos were cultured in vitro for 48 hours from the 8-cell to the blastocyst stage in media containing uniformly labelled ¹³C-glucose. The ¹³C content of the blastocysts was 20 atom % according to incorporation studies with ¹⁴C-glucose. No embryotoxic effects of carbon-13 incorporation could be detected on the basis of these criteria of normal development: the percentage of embryos reaching the blastocyst stage during the culture period; the number of cells in these blastocysts; and the development after transplantation to pseudopregnant foster mothers.     

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.     

NMR Spectroscopy of Cultured Astrocytes: Effects of Glutamine and the Gliotoxin Fluorocitrate

Abstract: Glial synthesis of glutamine, citrate, and other carbon skeletons, as well as metabolic effects of the gliotoxin fluorocitrate, were studied in cultured astrocytes with ¹³C and ³¹P NMR spectroscopy. f2–¹³C]Acetate and [1–¹³C]glucose were used as labeled precursors. In some experiments glutamine (2.5 mM) was added to the culture medium. Fluorocitrate (20 μM) inhibited the tricarboxylic acid (TCA) cycle without affecting the level of ATP. The net export of glutamine was reduced significantly, and that of citrate increased similarly, consistent with an inhibition of aconitase. Fluorocitrate (100 μM) inhibited TCA cycle activity even more and (without addition of glutamine) caused a 40% reduction in the level of ATP. In the presence of 2.5 mM glutamine, 100 μM fluorocitrate did not affect ATP levels, although glutamine synthesis was nearly fully blocked. The consumption of the added glutamine increased with increasing concentrations of fluorocitrate, whereas the consumption of glucose decreased. This shows that glutamine fed into the TCA cycle, substituting for glucose as an energy substrate. These findings may explain how fluorocitrate selectively lowers the level of glutamine and inhibits glutamine formation in the brain in vivo, viz., not by depleting glial cells of ATP, but by causing a rerouting of 2-oxoglutarate from glutamine synthesis into the TCA cycle during inhibition of aconitase. Analysis ^; of the ¹³C labeling of the C-2 versus the C-4 positions in glutamine obtained with [2–¹³C]acetate revealed that 57% of the TCA cycle intermediates were lost per turn of the cycle. Glutamine and citrate were the main TCA cycle intermediates to be released, but a large amount of lactate formed from TCA cycle intermediates was also released, showing that recycling of pyruvate takes place in astrocytes.     

Lactate: A major product of glutamine metabolism by human diploid fibroblasts

Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four μCi of glutamine-U-¹⁴C were added to media containing 5 mM or 65 μM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U¹⁴C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 μM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.     

Hormonal regulation of leucine catabolism in mammary epithelial cells

Branched-chain amino acids (BCAA) are actively taken up and catabolized by the mammary gland during lactation for syntheses of glutamate, glutamine and aspartate. Available evidence shows that the onset of lactation is associated with increases in circulating levels of cortisol, prolactin and glucagon, but decreases in insulin and growth hormone. This study determined the effects of physiological concentrations of these hormones on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 3 mM d-glucose, 0.5 mM l-leucine, l-[1-¹⁴C]leucine or l-[U-¹⁴C]leucine, and 0–50 μU/mL insulin, 0–20 ng/mL growth hormone 0–200 ng/mL prolactin, 0–150 nM cortisol or 0–300 pg/mL glucagon. Increasing extracellular concentrations of insulin did not affect leucine transamination or oxidative decarboxylation, but decreased the rate of oxidation of leucine carbons 2–6. Elevated levels of growth hormone dose dependently inhibited leucine catabolism, α-ketoisocaproate (KIC) production and the syntheses of glutamate plus glutamine. In contrast, cortisol and glucagon increased leucine transamination, leucine oxidative decarboxylation, KIC production, the oxidation of leucine 2–6 carbons and the syntheses of glutamate plus glutamine. Prolactin did not affect leucine catabolism in the cells. The changes in leucine degradation were consistent with alterations in abundances of BCAA transaminase and phosphorylated levels of branched-chain α-ketoacid dehydrogenase. Reductions in insulin and growth hormone but increases in cortisol and glucagon with lactation act in concert to stimulate BCAA catabolism for glutamate and glutamine syntheses. These coordinated changes in hormones may facilitate milk production in lactating mammals.     

Regulation and control of glucose overutilization in erythrocytes by vanadate

The insulin mimetic effect of vanadate inin vitro incubation of erythrocytes with high glucose concentrations showed an increase in sorbitol accumulation and glucose utilization using U-¹⁴C-glucose. Aldose reductase inhibitors and vanadate addition reversed the sorbitol accumulation, whereas insulin could not reverse it. Increased glucose utilization was also normalized with vanadium compounds. Increased activity of aldose reductase and sorbitol levels in diabetic animals were also normalized with vanadate treatment.     

Cultured rat hepatocytes adapt their cellular glycolytic activity and adenylate energy status to tissue oxygen tension: Influences of extracellular matrix components,insulin and glucagon

The influence of extracellular matrix components, insulin, and glucagon on the cellular response to periportal- or pericentral-equivalent tissue oxygen tension was investigated in freshly isolated rat hepatocytes cultured at 13% O₂ or 4% O₂ in Teflon membrane dishes. With extended culture time, significant increases in lactate release and cellular lactate content were observed in cultures at 4% O₂ compared with 13% O_2. This shift toward glycolysis was detectable when hepatocytes were cultured on dishes coated with rat liver crude membrane fraction (CMF/COL) but not in collagen type I-coated dishes. This indicates that extracellular matrix components are involved in the process of adaptation. ATP and total adenylate content in cells cultured at 4% O₂ were up to 40% lower than in cells cultured at 13% O_2. However, the adenylate energy charge was not affected, suggesting that an adequate energy supply was maintained also in hepatocytes cultured at pericentral-equivalent oxygen tension. This adaptation was reversible. When hepatocytes were transferred either from 4% to 13% O₂ or from 13% to 4% O₂, they adapted the corresponding metabolic profile to the new oxygen tension within 2 days. This demonstrates that hepatocytes are not fully unidirectionally programmed. The modulation of the glycolytic activity by insulin and glucagon was effective in cultures at pericentral-equivalent oxygen tension (4% O₂) only. Insulin (0.1-100 nM) counteracted the effect of insulin in a dose-dependent manner. Clearly, oxygen tension is the principal regulator in the hepatic glycolytic activity, whereas the hormones (insulin and glucagon) act as secondary modulators. © 1994 Wiley-Liss, Inc.     

Cultured trout liver cells: Utilization of substrates and response to hormones

Summary The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [¹⁴C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.     

Mechanisms of the fasting-induced dissociation of insulin binding from its action in isolated rat hepatocytes

Summary Fasting leads to an increase in insulin binding to isolated rat hepatocytes from 12 to 17%. This increase was accounted for by changes in the affinity of insulin receptors without alteration in their number. In contrast, the responsiveness of hepatocytes to insulin was markedly diminished in fasted rats. Both basal and insulin-stimulated rates of ¹⁴C-glucose incorporation into glycogen were significantly decreased in fasted animals. When insulin-induced ¹⁴C-glucose incorporation into glycogen was expressed as a percent above the basal rate, hepatocytes isolated both from control and fasted animals showed the same magnitude of maximal response (66 ± 13% in fed and 59 ± 12% in fasted animals, respectively). However, more insulin must be bound to hepatocytes isolated from fasted animals in order to elicit the same percent of insulin's maximal effect.Incubation of fed hepatocytes in the serum obtained from fasted rats significantly diminished their responsiveness to insulin. An addition of insulin (100 ng/ml), glucose (10 mM) and antibodies to glucagon (1:100) eliminated the inhibitory effect of fasted serum on fed hepatocytes.A 48-hour fast increased significantly the microviscosity (decreased fluidity) of hepatocyte plasma membranes and altered membrane phospholipid composition. These changes correlated with enhanced insulin binding to isolated membranes. Moreover, in response to insulin, plasma membranes isolated from fasted hepatocytes generated only one half the amount of the second messenger (PDH activator) observed in membranes of fed animals. The amount of PDH activator generated by incubation of plasma membranes with insulin correlated inversely with both insulin binding and membrane microviscosity.We conclude that 1) fasting induces both coupling defect and post-receptor changes in insulin's action; 2) both extracellular and intracellular factors contribute to fasting-induced dissociation of insulin binding from insulin action; 3) insulin/ glucagon ratio may influence hepatocyte responsiveness to insulin; 4) alterations in plasma membrane fluidity and phospholipid composition may alter insulin binding and contribute to its dissociation from the subsequent action; 5) membranes isolated from fasted hepatocytes generate less mediator of insulin action than do membranes isolated from fed hepatocytes.This work was presented in part at the Meetings of the Western Society of the American Federal for Clinical Research, February 17, 1982, Carmel, California and February 9, 1983, Carmel, California.